Our recent work provided unexpected but convincing evidence that 3H-prostaglandins (PG) E1 and F2Alpha, bind (specific) not only to plasma membranes (PM) but also to nuclear membranes (NM), lysosomes (LY), rought endoplasmic reticulum (RER) and three (light, medium and heavy) subfractions of golgi (GFs) of bovine corpora lutea. Some of the intracellular binding sites undoubtedly represent those in the biosynthetic (RER & GFs) and degradative (LY) pathways, while the others (NM) do not fit into this scheme. Even for RER, GFs, and LY binding sites, some functional significance, in addition to biosynthesis and catabolism, cannot be ruled out. The presence of intracellular binding sites for PGE1 and PGF2Alpha raises three very important questions a) What are the properties of intracellular binding sites and how do they compare with those of PM? b) Do PGE1 and PGF2Alpha enter luteal cells and if so, do they bind to some or all of these intracelluar organelles? c) Does intracellular binding represent only biosynthesis and catabolism and/or is there some functional significance to this? The aim of this grant proposal is to seek answers to these questions. The luteal tissue from cows and superovulated rats will be used because they provide flexibility and convenience in accomplishing our goals. Purified PM, NM, LY, RER, and GFs will be prepared and their purity assessed by marker enzymes and electron microscopy (EM). These fractions will be used in studying binding properties, PGE1 and PGF2Alpha effects on functions established as normal to these organelles, receptor profiles during luteal growth and demise and during down regulation and recovery phase induced by injecting 16, 16-dimethyl PGE1 and PGF2Alpha. The combination of indirect (binding) and direct autoradiography-EM) approaches will be used to evaluate cellular entry of PGE1 and PGF2Alpha. In summary, the studies outlined in the proposal are very important and concept changing not only for PGE1 and PGF2Alpha action but may also be applicable to other agents believed to bind and act through PM receptors exclusively.